青花椒雄蕊调控基因ZaPI的克隆、鉴定与表达模式分析

Cloning,Characterization and Expression Pattern Analysis of the Cloning,Identificatoin and Expression Pattern Analysis of the Stamen Regulatory Gene ZaPI in Zanthoxylum armatum DC.

【目的】探究青花椒雄蕊分化关键B类基因及其核酸和蛋白序列特性,并分析其在雌雄花分化过程的表达模式,为青花椒花芽性别分化的遗传调控机制提供候选基因和数据支撑。【方法】基于青花椒转录组和基因组数据库,提取雌雄花分化过程中差异表达的所有B类MADS-box家族基因,并分析获取关键成员;克隆关键成员基因编码序列,全面剖析核酸和蛋白序列特性;并解析其在11种不同组织器官以及雌雄花分化过程的表达模式。【结果】青花椒雌雄花分化过程的差异基因中共有11个B类MADS-box家族基因,其中Zardc17043(ZaPI)的表达量最高,且在雄花中极显著特异表达。该ZaPI编码序列全长624 bp,共编码207个氨基酸,含有77个氨基酸组成的MADS-box结构域和84个氨基酸组成的K-box结构域,蛋白相对分子量为24.29 kD,是位于11号染色体包含4个外显子和3个内含子且定位于细胞核的亲水性蛋白;系统进化分析显示,青花椒ZaPI与同为芸香科(Rutaceae)的克莱门柚(Citrus clementina)和甜橙(Citrus sinensis)亲缘关系最近。此外,青花椒ZaPI基因编码序列共预测到54个转录因子结合位点,主要涉及植物生殖生长和抗逆过程等多种调控途径。顺式作用元件分析发现,ZaPI启动子区富含光信号、植物激素、逆境胁迫等相关的顺式作用元件。同时,该基因在根茎叶刺等营养器官中几乎不表达,雌花分化过程的表达量也相对较低,但在雄花分化过程中呈极显著上调趋势。【结论】研究所获得的ZaPI基因与雄蕊的分化显著相关,可能与植物激素等多种信号分子互作参与了青花椒的雄蕊形成过程。研究所获结果对于进一步探究青花椒雄蕊形成的分子机制,培育高产青花椒种质资源提供了丰富的理论支撑。

【Objective】To investigate the key B-class genes involved in stamen differentiation in Sichuan pepper Zanthoxylum armatum DC.,along with their nucleic acid and protein characteristics,and analyze their expression patterns during male and female flower differentiation processes,in order to provide candidate genes and data support for the genetic regulatory mechanisms of floral bud sex differentiation in Sichuan pepper.【Method】Based on the transcriptome and genome databases of Sichuan pepper,all differentially expressed B-class MADS-box family genes during male and female flower differentiation were extracted,and key members were identified.The gene coding sequences of these key members were cloned to comprehensively analyze their nucleic acid and protein sequence characteristics.Furthermore,their expression patterns were analyzed in 11 different tissues and organs,as well as in the differentiation processes of male and female flower.【Result】Among the differentially expressed genes during the differentiation of male and female flowers in Sichuan pepper,there were a total of 11 B-class MADS-box family genes.Among them,Zardc17043(ZaPI)exhibited the highest expression level and showed highly significant specific expression in male flowers.The results revealed that the coding sequence of ZaPI is 624 bp,encoding 207 amino acids,including a MADS-box domain(77 amino acids)and a Kbox domain(84 amino acids).The relative molecular weight of the protein is 24.29 kD.Phylogenetic analysis indicated that ZaPI was most closely related to Citrus clementina and Citrus sinensis,both from the Rutaceae family.In addition,a total of 54 transcription factor binding sites were predicted in the coding sequence of the ZaPI gene of Sichuan pepper,which were primarily involved in various regulatory pathways,such as plant reproductive growth and stress resistance processes.Cis-acting element analysis revealed that the ZaPI promoter region was rich in those cis-acting elements related to light signaling,phytohormones,and adversity stress.Meanwhile,ZaPI was minimal expressed in nutritional organs such as rootstocks and leaf spines,and its expression was relatively low during female flower differentiation,but it exhibited significant up-regulation during male flower differentiation,suggesting its potential involvement in the differentiation process of green pepper stamens.【Conclusion】The ZaPI gene obtained in this study is significantly associated with the differentiation of stamens and may be involved in the process of stamen formation in Sichuan pepper through interactions with various signaling molecules,including plant hormones.The results provide rich theoretical support for further exploration of the molecular mechanisms underlying stamen formation in Sichuan pepper and the cultivation of high-yielding germplasm resources.